EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785

Abstract

Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked βGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression.

Figure 1

Transmission electron microscopy of wild type and mutant strains. L . johnsonii FI9785 (wild type) and its derivatives ΔepsA, made by deletion mutagenesis using the thermosensitive pG⁺host9 vector system, and complemented strain ΔepsA::pepsA (see supporting information) were grown and visualized as described previously (Dertli et al., 2013). The bar represents 100 nm. CW, cell wall, PM, plasma membrane.

Figure 2

Analysis of EPS production and quality. A. Structure of the EPS‐1 and EPS‐2 produced by L . johnsonii FI9785 showing labelled sugar residues b, c from EPS‐1 and residues a and d–h from EPS‐2 (Dertli et al., 2013). B. 600 MHz ¹H NMR spectrum (338°K, D₂O) of EPS extracted from the ΔepsA::pepsA mutant. EPS were isolated from 500 ml cultures as previously described (Tallon et al., 2003); lyophilized samples were dissolved in D₂O and 1D and 2D NMR spectra were obtained using the same pulse sequences as described previously (Dertli et al., 2013). C. Growth of strains to stationary phase. Optical density (OD600) readings represent the mean of triplicate samples +/− standard deviation. ■, wild type; □, ΔepsA; ▲, ΔepsA::pepsA. D. Increase in EPS production after complementation. EPS were quantified using the phenol‐sulfuric acid method (DuBois et al., 1956) with glucose as a standard and expressed as a quantity of EPS production per 10⁷ cfu (mean of triplicate samples +/− standard deviation). Black, wild type; white, ΔepsA::pepsA. E. Aggregation of wild type and mutant strains. The aggregation percentage was measured after overnight incubation (16 h) by flow cytometry (Dertli et al., 2015). Results are the mean of triplicates +/− standard deviation. Black, wild type; grey, ΔepsA; white, ΔepsA::pepsA.